Project Summary Carbohydrate chains, known as glycans, are fundamental to a wide spectrum of biological processes, including anticoagulation, cell growth and development, cell-cell communication, immune recognition/response, and microbial pathogenesis. Glycans also feature prominently in human disease. For example, glycans are commonly expressed at atypical levels on tumor cells and have been shown to regulate tumor proliferation, invasion, hematogenous metastasis, and angiogenesis. A better understanding of the role that glycans play in both health and disease could be achieved with a comprehensive set of affinity reagents that specifically recognize defined glycoforms. Unfortunately, the number of commercially available glycan-binding proteins such as antibodies or lectins is greatly eclipsed by the number of biologically important glycan epitopes (glycotopes), and there are no commercially available antibodies specific for N-glycan structures. One approach with the potential to bridge this technology deficit is the isolation of antibodies with exquisite specificity for distinct glycans or glycoconjugates such as glycoproteins and glycolipids. However, obtaining antibodies that recognize specific glycoforms can be challenging for a number of technical reasons, thereby limiting the pace at which new orthogonal antibody-glycotope pairs are developed. To address these challenges, the objective of this proposal is to create a robust, integrated pipeline for producing renewable, well-defined glycotopes and corresponding high-affinity glycan-recognizing antibodies (grAbs) in a rapid and routine manner. Under Specific Aim 1, specially engineered Escherichia coli cells along with a new bioenzymatic synthesis strategy will be used to produce 4 structurally uniform free reducing-end N-glycan structures. The resulting glycans will be used for direct selection and characterization of grAbs. A scalable method for discovering synthetic antibody fragments from phage-displayed combinatorial libraries will be adapted for parallel in vitro selections of antibodies against immobilized E. coli-derived glycotopes. Under Specific Aim 2, all isolated grAbs will be characterized using ELISA, surface plasmon resonance, and glycan microarrays to determine affinity and specificity for the targeted glycan structure. In addition, grAbs will be used to probe the temporal and spatial expression of authentic glycan signatures on the surfaces of cell lines or in the context of glycoproteins. Given its modularity and scalability, this grAb-glycotope pipeline has the potential to yield customized affinity reagents on a glycome-wide scale, providing a high-quality and easy to use toolkit for glycan analysis that will be broadly accessible to the laboratories of specialists and non-specialists alike.